Methods and compositions for increasing the anaerobic working in tissues

ABSTRACT

A method for increasing the synthesis and accumulation of beta-alanylhistidine dipeptides, with a simultaneous increase in the accumulation of creatine, in bodily tissues of humans and animals is described. This is accomplished by causing an increase in the blood plasma concentrations of beta-alanine and creatine, or the blood plasma concentrations of beta-alanine, L-histidine and creatine, by the ingestion or infusion of a composition including beta-alanine, beta-alanine and creatine, or beta-alanine, L-histidine and creatine, or active derivatives thereof.

This is a divisional of U.S. application Ser. No. 08/909,513, filed Aug.12, 1997, now U.S. Pat. No. 5,965,596.

BACKGROUND OF THE INVENTION

The invention relates to methods and compositions for increasing theanaerobic working capacity of muscle and other tissues.

Natural food supplements are typically designed to compensate forreduced levels of nutrients in the modern human and animal diet. Inparticular, useful supplements increase the function of tissues whenconsumed. It can be particularly important to supplement the diets ofparticular classes of animals whose the normal diet may be deficient innutrients available only from meat and animal produce (e.g., humanvegetarians and other animals consume an herbivorous diet).

For example, in the sporting and athletic community, natural foodsupplements which specifically improve athletic ability are increasinglyimportant, such as supplements that promote or enhance physical prowessfor leisure or employment purposes. In another example, anaerobic (e.g.,lactate-producing) stress can cause the onset of fatigue and discomfortthat can be experienced with aging. Anaerobic stress can also resultfrom prolonged submaximal isometric exercise when the local circulationis partially or totally occluded by the increase in intra-muscularpressure (e.g., during rock climbing, free diving, or synchronizedswimming). Excessive lactate production can result in the acidificationof the intracellular environment.

Creatine (i.e., N-(aminoiminomethyl)-N-glycine, N-amidinosarcosine,N-methyl-N-guanylglycine, or methylglycocyamine) is found in largeamounts in skeletal muscle and other “excitable” tissues (e.g., smoothmuscle, cardiac muscle, or spermatozoa) characterized by a capacity fora high and variable energy demand. Creatine is converted intophosphorylcreatine in energy-generating biochemical pathways withincells. In mammalian skeletal muscle, the typical combined content ofcreatine (i.e., creatine and phosphorylcreatine) may vary from less than25 to about 50 mmol per kilogram fresh muscle (i.e., 3.2 to 6.5 gramsper kilogram fresh muscle).

Creatine formed is formed in the liver and taken up into tissues, suchas muscle, by means of an active transport system. Creatine synthesis inthe body may also be augmented by the ingestion of creatine present inmeat (e.g., 5-10 milligrams per kilogram body weight per day in theaverage meat-eating human and approximately zero in a vegetarian diet).

During sustained intensive exercise, or exercise sustained underconditions of local hypoxia, the accumulation of hydronium ions formedduring glycolysis and the accumulation of lactate (anaerobic metabolism)can severely reduce the intracellular pH. The reduced pH can compromisethe function of the creatine-phosphorylcreatine system. The decline inintracellular pH can affect other functions within the cells, such asthe function of the contractile proteins in muscle fibers.

Dipeptides of beta-alanine and histidine, and their methylatedanalogues, include carnosine (beta-alanyl-L-histidine), anserine(beta-alanyl-L-1-methylhistidine), or balenine(beta-alanyl-L-3-methylhistidine). The dipeptides are present in themuscles of humans and other vertebrates. Carnosine is found inappreciable amounts in muscle of, for example, humans and equines.Anserine and carnosine are found in muscle of, for example, canines,camelids and numerous avian species. Anserine is the predominantbeta-alanylhistidine dipeptide in many fish. Balenine is the predominantbeta-alanylhistidine dipeptide in some species of aquatic mammals andreptiles. In humans, equines, and camelids, the highest concentrationsof the beta-alanylhistidine dipeptides are found in fast-contractingglycolytic muscle fibers (type IIA and IIB) which are used extensivelyduring intense exercise. Lower concentrations are found in oxidativeslow-contracting muscle fibers (type I). See, e.g., Dunnett, M. &Harris, R. C. Equine Vet. J., Suppl. 18, 214-217 (1995). It has beenestimated that carnosine contributes to hydronium ion buffering capacityin different muscle fiber types; up to 50% of the total in equine typeII fibers.

SUMMARY OF THE INVENTION

In general, the invention features methods and compositions forincreasing the anaerobic working capacity of muscle and other tissues.The method includes simultaneous accumulation of creatine andbeta-alanylhistidine dipeptides, or beta-alanine and L-histidineanalogues, within a tissue in the body. The methods include ingesting orinfusing compositions into the body. The compositions are mixtures ofcompounds capable of increasing the availability and uptake of creatineand of precursors for the synthesis and accumulation ofbeta-alanylhistidine dipeptides, in human and animal muscle. Thecomposition induces the synthesis and accumulation ofbeta-alanylhistidine dipeptides in a human or animal body whenintroduced into the body.

The compositions include mixtures of creatine and beta-alanine,creatine, beta-alanine and L-histidine, or creatine and activederivatives of beta-alanine or L-histidine. Each of the beta-alanine orL-histidine can be the individual amino acids, or components ofdipeptides, oligopeptides, or polypeptides. The beta-alanine orL-histidine can be active derivatives. An active derivative is acompound derived from, or a precursor of, the substance that performs inthe same or similar way in the body as the substance, or which isprocessed into the substance and placed into the body. Examples include,for example, esters and amides.

In one aspect, the invention features a method of regulating hydroniumion concentrations in a tissue. The method includes the steps ofproviding an amount of beta-alanine to blood or blood plasma effectiveto increase beta-alanylhistidine dipeptide synthesis in a tissue, andexposing the tissue to the blood or blood plasma, whereby theconcentration of beta-alanylhistidine is increased in the tissue. Themethod can include the step of providing an amount of L-histidine to theblood or blood plasma effective to increase beta-alanylhistidinedipeptide synthesis.

In another aspect, the invention features a method of increasing theanaerobic working capacity of a tissue. The method includes the steps ofproviding an amount of beta-alanine to blood or blood plasma effectiveto increase beta-alanylhistidine dipeptide synthesis in a tissue,providing an amount of L-histidine to the blood or blood plasmaeffective to increase beta-alanylhistidine dipeptide synthesis in atissue, and exposing the tissue to the blood or blood plasma. Theconcentration of beta-alanylhistidine is increased in the tissue.

In embodiments, the methods can include the step of increasing aconcentration of creatine in the tissue. The increasing step can includeproviding an amount of creatine to the blood or blood plasma effectiveto increase the concentration of creatine in the tissue (e.g., byproviding the amount of creatine to the blood or blood plasma).

The providing steps of the methods can include ingestion or infusion(e.g., injection) of a composition including the amount of beta-alanine,or the amounts of beta-alanine and L-histidine, or a combination ofingestion and infusion.

The methods can include increasing a concentration of insulin in theblood or blood plasma. The concentration of insulin can be increased,for example, by injection of insulin.

The tissue can be a skeletal muscle.

In another aspect, the invention features a composition consistingessentially of a peptide source including beta-alanine, between about 39and about 99 percent by weight of a carbohydrate, and up to about 60percent by weight of water. The composition includes between about 1 andabout 20 percent by weight of the beta-alanine. The peptide source caninclude L-histidine. The composition can include between about 1 andabout 20 percent by weight of L-histidine.

The carbohydrate can be a simple carbohydrate (e.g., glucose). Inanother aspect, the invention features a composition consistingessentially of a peptide source including beta-alanine, between about 1and about 98 percent by weight of a creatine source, and up to about 97percent by weight of water. The composition includes between about 1 andabout 98 percent by weight of the beta-alanine. The peptide source caninclude L-histidine and the composition includes between about 1 andabout 98 percent by weight of L-histidine.

The peptide source can be a mixture of amino acids, dipeptides,oligopeptides, polypeptides, or active derivatives thereof.

The composition can be a dietary supplement. The creatine source can becreatine monohydrate.

The concentrations of components in blood or blood plasma can beincreased by infusion (i.e., injection) or ingestion of an agentoperable to cause an increase in the blood plasma concentration. Thecomposition can be ingested in doses of between about 10 grams and about800 grams per day. The doses can be administered in one part or multipleparts each day.

An increase in the muscle content of creatine and beta-alanylhistidinedipeptides can increase the tolerance of the cells to the increase inhydronium ion production with anaerobic work, and to lead to an increasein the duration of the exercise before the onset of fatigue. Thecompositions and methods can contribute to correcting the loss ofbeta-alanine, L-histidine, or creatine due to degradation or leaching ofthese constituents during cooking or processing. The compositions andmethods can also contribute to correcting the absence of thesecomponents from a vegetarian diet.

The methods and compositions can be used to increasebeta-alanylhistidine dipeptide by, for example, sportsmen, athletes,body-builders, synchronized swimmers, soldiers, elderly people, horsesin competition, working and racing dogs, and game birds, to avoid ordelay the onset of muscular fatigue.

Other advantages and features of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting changes in the concentrations ofbeta-alanine in blood plasma of five horses, before and at 2 hourintervals following the feeding of beta-alanine and L-histidine (100milligrams per kilogram body weight and 12.5 milligrams per kilogrambody weight, respectively, three times per day) over a period of 30days.

FIG. 2 is a graph depicting changes in the concentrations of L-histidinein blood plasma of five horses, before and at 2 hour intervals followingthe feeding of beta-alanine and L-histidine (100 milligrams per kilogrambody weight and 12.5 milligrams per kilogram body weight, respectively,three times per day) over a period of 30 days.

FIGS. 3 a and 3 b are graphs depicting the contrast in the changes inthe concentrations of beta-alanine in blood plasma of six horses, beforeand at hourly intervals following the feeding of beta-alanine andL-histidine (100 milligrams per kilogram body weight and 12.5 milligramsper kilogram body weight, respectively, three times per day) on thefirst and last day of a 30 day period of dietary supplementation.

FIGS. 4 a and 4 b are graphs depicting the contrast in the changes inthe concentrations of L-histidine in blood plasma of six horses, beforeand at hourly intervals following the feeding of beta-alanine andL-histidine (100 milligrams per kilogram body weight and 12.5 milligramsper kilogram body weight, respectively, three times per day) on thefirst and last day of a 30 day period of dietary supplementation.

FIG. 5 is a graph depicting the contrast in the changes in the meanconcentrations of beta-alanine in equine blood plasma (n=6), before andat hourly intervals following the feeding of beta-alanine andL-histidine (100 milligrams per kilogram body weight and 12.5 milligramsper kilogram body weight, respectively, three times per day) on thefirst and last day of a 30 day period of dietary supplementation.

FIG. 6 is a graph depicting the contrast in the changes in the meanconcentrations of L-histidine in equine blood plasma (n=6), before andat hourly intervals following the feeding of beta-alanine andL-histidine (100 milligrams per kilogram body weight and 12.5 milligramsper kilogram body weight, respectively, three times per day) on thefirst and last day of a 30 day period of dietary supplementation.

FIG. 7 is a graph depicting the correlation between the increase in 6thoroughbred horses in the carnosine concentration in type II skeletalmuscle fibers (the average of the sum of type IIA and IIB fibres) andthe increase, between the 1st and 30th day of supplementation, in thearea under the blood plasma beta-alanine concentration-time curve overthe first 12 hours of the day (AUC_((0-12 hr)))

FIG. 8 is graph depicting the mean results of the administration ofbeta-alanine, broth, or carnosine to test subjects.

FIG. 9 is a graph depicting mean changes in plasma beta-alanine overnine hours of treatment.

FIG. 10 is a graph depicting the mean changes in plasma beta-alanineover 9 hours following the oral ingestion of 10 milligrams per kilogrambody weight of beta-alanine.

FIG. 11 is a graph depicting the mean (n=6) plasma beta-alanineconcentration over the 24 hour of Day 1 and Day 30 of the treatmentperiod.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Beta-alanylhistidine dipeptides such as carnosine, anserine, andbalenine have pKa values between approximately 6.8 and 7.1 and areinvolved in the regulation of intra-cellular pH homeostasis duringmuscle contraction and the development of fatigue. The content of othersubstances involved in hydronium ion buffering, such as amino acidresidues in proteins, inorganic and organic phosphates and bicarbonate,is constrained by their involvement in other cell functions. Thebeta-alanylhistidine dipeptides can provide an effective way ofaccumulating pH-sensitive histidine residues into a cell. Variations inthe muscle beta-alanylhistidine dipeptide concentrations affect theanaerobic work capacity of individual athletes.

The beta-alanylhistidine dipeptides are synthesized within the body frombeta-alanine and L-histidine. These precursors can be generated withinthe body or are made available via the diet, including from thebreakdown of an ingested beta-alanylhistidine dipeptide. Beta-alaninewithin the body is transported to tissues such as muscle. In a typicalfed state, the concentration of beta-alanine is low in comparison withthe concentration of L-histidine in human and equine blood plasma. Theseconcentrations should be viewed in relation to the affinity of thecarnosine synthesizing enzyme, carnosine synthetase, for its substratesas determined by the Michaelis-Menton constant (Km). The Km forhistidine is about 16.8 μM. The Km for beta-alanine is between about1000 and 2300 μM. The low affinity of carnosine synthetase forbeta-alanine, and the low concentration of beta-alanine in muscle,demonstrate that the concentration of beta-alanine in muscle which islimiting to the synthesis of the beta-alanylhistidine dipeptides.

Increasing the amount of beta-alanylhistidine dipeptides within a musclefavorably affects muscular performance and the amount of work that canbe performed by the muscle. Accordingly, the synthesis and accumulationof beta-alanylhistidine dipeptides is increased in a tissue in a humanor animal body.

The synthesis and accumulation of beta-alanylhistidine dipeptides in ahuman or animal body can be increased with an increase in the contentwithin the body of creatine, by increasing the blood or blood plasmaconcentrations of beta-alanine, increasing the blood or blood plasmaconcentrations beta-alanine and creatine, or increasing the blood orblood plasma concentrations beta-alanine, L-histidine, and creatine. Theincrease in dipeptide can be simultaneous with the increase inbeta-alanine concentration.

The blood plasma concentrations of beta-alanine, L-histidine andcreatine can be increased by ingestion or infusion of beta-alanine,L-histidine, and creatine, or active derivatives thereof. Thecomposition can be administered orally, enterally, or parenterally. Thebeta-alanine and creatine, or beta-alanine, L-histidine and creatine,are preferably orally ingested.

The composition can include carbohydrates (e.g., simple carbohydrates),insulin, or agents that stimulate the production of insulin.

The composition can be ingested as a dietary supplement. Preferably, thecomposition can be administered in one or more doses per day. Thebeta-alanine dosage can be between about 5 milligrams and about 200milligrams per kilogram body weight. The creatine (e.g., creatinemonohydrate) dosage can be between about 5 milligrams to 200 milligramsper kilogram body weight. The L-histidine dosage can be between about 1milligrams to 100 milligrams per kilogram body weight. The simplecarbohydrate (e.g., glucose) dosage can be between about 0.5 and 2.0grams per kilogram body weight.

In an 80 kilogram person, suitable dosages per day can be between 0.4grams to 16.0 grams of beta-alanine, 0.4 grams to 16.0 grams of creatinemonohydrate, 0.08 grams to 8.0 grams of L-histidine, or 40 grams to 160grams of glucose or other simple carbohydrate. The composition can be insolid form or liquid form or the form of a suspension which is ingested,or in liquid form or suspension for infusion into the body. Thecomposition is ingested in humans in an amount of between 2 grams and1000 grams per day (e.g., between 10 grams and 800 grams), which may betaken in one or more parts throughout the day. In animals the dailyintake will be adjusted for body weight.

For humans and animals, the compositions can be:

(a) 1% to 99% by weight of beta-alanine;

 1% to 99% by weight of creatine monohydrate; and

 0% to 98% by weight of water;

(b) 1% to 98% by weight of beta-alanine;

 1% to 98% by weight of L-histidine;

 1% to 98% by weight of creatine monohydrate; and

 0% to 97% by weight of water;

(c) 1% to 20% by weight of beta-alanine;

 39% to 99% by weight of glucose or other simple carbohydrate; and

 0% to 60% by weight of water; or

(d) 1% to 20% by weight of beta-alanine;

 1% to 20% by weight of L-histidine

 39% to 99% by weight of glucose or other simple carbohydrate; and

 0% to 60% by weight of water.

The following are specific examples of the methods of methods andcompositions for increasing the anaerobic working capacity of muscle andother tissues.

EXAMPLE 1

The effect of supplementation of a normal diet with multiple daily dosesof beta-alanine and L-histidine on the carnosine concentration in typeI, IIA, and IIB skeletal muscle fibers of thoroughbred horses wasassessed. Six experimental thoroughbred horses of normal health (threefillies and three geldings), aged 4 to 9 years, underwent one month (30days) of dietary conditioning (pre-supplementation period) prior to thecommencement of the supplementation period. During the dietaryconditioning period each horse was fed a diet comprising 1 kilogram ofpelleted feed (Spillers racehorse cubes) and 1 kilogram of soaked sugarbeet pulp as a source of complex and simple carbohydrates, three timesper day (at 08:30, 12:30, and 16:30, respectively). Soaked hay (3kilograms dry weight) was also provided twice daily (at 09:00 and17:00). Water was provided ad libitum.

During the supplementation period an identical feeding regime wasimplemented. However, each hard feed meal was supplemented withbeta-alanine and L-histidine (free base). Beta-alanine and L-histidinewere mixed directly into the normal feed. Individual doses ofbeta-alanine and L-histidine were calculated according to body weight.Beta-alanine was administered at 100 milligrams per kilogram body weightand L-histidine at 12.5 milligrams per kilogram body weight. Dietarysupplementation was begun on day 1 of the protocol and discontinued atthe end of day 30. Heparinized blood samples (5 milliliters) werecollected on days 1, 6, 18, 24, and 30. On day 1 and day 30, bloodsamples were collected prior to the first feed and at hourly intervalsfor a total of 12 hours each day. On the three intervening samplingdays, blood was collected prior to the first feed and 2 hours after eachsubsequent feed. On the day before the start of supplementation (day 0)a muscle biopsy was taken, following application of local anaesthesia ofthe skin, from the right middle gluteal muscle (m. gluteus medius) ofeach horse using a Bergstrom-Stille percutaneous biopsy needle.Subsequent muscle biopsies were collected immediately after the end ofthe supplementation period (day 31) as close as possible to the originalsampling site. Clinical monitoring of the horses was performed daily.This comprised a visual examination and measurement of body weight,twice-daily measurement of rectal temperature, and weekly blood samplingfor clinical biochemistry and hematology. During the course of the studythe horses received no formal training or exercise, although they wereallowed one hour of free exercise each day.

Fragments of individual muscle fibers dissected from freeze-dried musclebiopsies were characterized as either type I, IIA or IIB byhistochemical staining for myosin ATPase activity at pH 9.6 followingpre-incubation at pH 4.50 by a modification of the method described in,Kaiser and Brook, Arch. Neurol., 23:369-379 (1970).

Heparinized blood plasma samples were extracted and analyzed forbeta-alanine and L-histidine concentrations by high-performance liquidchromatography (HPLC). Individual weighed muscle fibers were extractedand analyzed for carnosine by HPLC according to the method described in,Dunnett and Harris, “High-performance liquid chromatographicdetermination of imidazole dipeptides, histidine, 1-methylhistidine and3-methylhistidine in muscle and individual muscle fibers,” J.Chromatogr. B. Biomed. Appl., 688:47-55 (1997).

Differences in carnosine concentrations within fiber types before andafter supplementation were established within horses using one-wayanalysis of variance (ANOVA). In instances where differences weredetected, significance was determined using a multiple comparison test(Fisher's PLSD).

No palatability problems were encountered with the addition ofbeta-alanine and L-histidine to the feed. No adverse physiological orbehavioral effects of the supplemented diet were observed in any of thehorses during the thirty days of supplementation. No significant changesin body weight were recorded, and rectal temperatures remained withinthe normal range. No acute or chronic changes in clinical biochemistryor hematology were observed. Beta-alanine was not detected in the plasmaof any of the horses prior to the start of supplementation. The lowerlimit of quantitation for beta-alanine in plasma by the assay used was 3micromolar (μM). Plasma L-histidine concentrations in the six horsesprior to the start of supplementation were between 36.6 and 54.4 μM.

Individual changes in blood plasma beta-alanine and L-histidineconcentrations for five of the six horses over on all the sampling daysare shown in FIGS. 1 and 2, respectively. There was a trend towards anincrease in the pre-feeding concentrations of blood plasma beta-alanineand L-histidine with increasing time of supplementation. Furthermore,over the thirty day supplementation period, the blood plasmaconcentration response to supplementation was also increased. Theresponse was greater for beta-alanine.

Comparisons of the changes in blood plasma beta-alanine and L-histidineconcentrations prior to the first feed of the day, and hourly thereafterbetween the first and last days of the supplementation period, for thesix individual horses, are shown in FIGS. 3 a and 3 b, and FIGS. 4 a and4 b, respectively. The mean (±SD) changes (n=6) in blood plasmabeta-alanine concentration over time during the 24 hours of the first(day 1) and last (day 30) days of the supplementation period arecontrasted in FIG. 5. The area under the mean blood plasma beta-alanineconcentration versus time curve over 24 hours (AUC_((0-24 hr))) was muchgreater on day 30 of the supplementation.

The mean (±SD) changes (n=6) in blood plasma L-histidine concentrationover time during the 24 hours of the first (day 1) and last (day 30)days of the supplementation period are contrasted in FIG. 6. The areaunder the mean blood plasma beta-alanine concentration vs. time curveover 24 hours (AUC_((0-24 hr))) was greater on day 30 of thesupplementation. The greater AUC for blood plasma beta-alanine on thelast day of supplementation (day 30) in contrast to the first day ofsupplementation (day 1) suggests the increased uptake of beta-alaninefrom the equine gastro-intestinal tract with progressivesupplementation. A similar effect was observed for changes in bloodplasma L-histidine concentration during the supplementation period. Peakblood plasma concentrations of beta-alanine and L-histidine occurredapproximately one to two hours post-feeding in each case.

A total of 397 individual skeletal muscle fibers (192pre-supplementation; 205 post-supplementation) from the six horses weredissected and analyzed for carnosine. Mean (±SD) carnosineconcentration, expressed as millimoles per kilogram dry weight (mmolkg⁻¹ dw), in pre- and post-supplementation type I, IIA, and IIB skeletalmuscle fibers from the six individual horses are given in Table 1 wheren is the number of individual muscle fibers analyzed. Following thirtydays of beta-alanine and L-histidine supplementation the mean carnosineconcentration was increased in type IIA and IIB fibers in all sixhorses. These increases were statistically significant in seveninstances. The increase in mean carnosine concentration in type IIBskeletal muscle fibers was statistically significant in five out of sixhorses. The increase in mean carnosine concentration in type IIAskeletal muscle fibers was statistically significant in two out of sixhorses.

TABLE 1 Horse Day Type 1 n Type IIA n Type IIB n 6 0 32.3 3 72.1 11111.8 14 31 (14.5) (47.7) 17 (22.8) 12 — 16.2 117.7 (20.9) (38.7) 5 059.5 2 102.6 12 131.2 26 31 (3.9) 1 (12.7) 18 (26.6) 22 55.5 112.2 153.3(17.1) (28.0)** 4 0 44.8 4 59.9 13 108.6 19 31 (6.6) 2 (19.5) 17 (41.5)19 37.0 88.0 152.4 (9.3) (34.2)* (65.0)* 1 0 56.7 2 88.5 15 101.3 13 31(5.3) 1 (20.9) 19 (15.2) 11 57.8 96.1 14.3 (17.3) (13.3)* 2 0 — 89.6 13104.2 14 31 65.9 4 (16.2) 18 (22.2) 12 (13.2) 102.2 142.0 (22.1)(35.4)*** 3 0 30.9 2 85.1 6 113.5 23 31 (4.0) (20.3) 23 (20.4) 9 — 105.0135.4 (17.6)* (24.9)* Mean 0 44.8 13 83.0 70 111.8 109 31 54.1 8 96.6*112 135.9** 85 *significantly different to pre-supplementation, p < 0.05**significantly different to pre-supplementation, p < 0.01***significantly different to pre-supplementation, p < 0.005

The absolute (e.g. mmol kg⁻¹ dw) and percentage increases the meancarnosine concentrations in type IIA and IIB skeletal muscle fibers fromthe six horses are listed in Table 2.

TABLE 2 Type IIA Type IIA Type IIB Type IIB Horse Absolute increase %increase Absolute increase % increase 6  4.1  5.7  5.6  5.3 5  9.6  9.422.1 16.8 4 28.1 46.9 43.8 40.3 1  7.6  8.6 13.0 12.8 2 12.6 14.1 37.836.3 3 19.9 23.4 21.9 19.3 Mean 13.6 18.0 24.1 21.8

It was observed that the individual horses which showed the greaterincrease in muscle carnosine concentration following thirty days ofsupplementation also demonstrated the greater increase in blood plasmabeta-alanine AUC between day 1 and day 30 of the supplementation period.Referring to FIG. 7, a significant correlation (r=0.986, p<0.005) forfive of the six horses was observed between the increase in meancarnosine concentration, averaged between type IIA and IIB skeletalmuscle fibers and the increase, between the 1st and 30th day ofsupplementation, in blood plasma beta-alanine AUC, over the first 12hours(AUC_((0-12 hr))). Only five horses were used to calculate theregression line. Horse 6 (filled circle) showed no appreciable increasein blood plasma beta-alanine concentration greater than that observed onday 1 until the last day of supplementation. This was unlike the otherfive horses which showed a progressive increase with each sampling day.For this reason horse 6 was excluded from the calculation of theregression equation.

Increases in muscle carnosine concentration following thirty days ofsupplementation with beta-alanine and L-histidine will cause a directincrease in total muscle buffering capacity. This increase can becalculated by using the Henderson-Hasselbach Equation. Calculated valuesfor the increases in muscle buffering capacity in type IIA and IIBskeletal muscle fibers in the six thoroughbred horses are shown in Table3.

TABLE 3 Type Type Type IIA Type Type Type IIB IIA IIA Δβmtotal IIB IIBΔβmtotal Horse Day βmcar βmtotal (%) βmcar βmtotal (%) 6 0 23.9 93.937.1 107.1 31 25.3 95.3 +1.5 39.0 109.0 +1.8 5 0 34.0 104.0 43.5 113.531 37.2 107.2 +3.1 50.8 120.8 +6.4 4 0 19.9 89.9 36.0 106.0 31 29.2 99.2+10.3 50.5 120.5 +13.7 1 0 29.3 99.3 33.6 103.6 31 31.9 101.9 +2.6 37.9107.9 +4.2 2 0 29.7 99.7 34.5 104.5 +12.1 31 33.9 103.9 +4.2 47.1 117.13 0 28.2 98.2 37.6 107.6 31 34.8 104.8 +6.7 44.9 114.9 +6.8 Mean 0 27.597.5 37.1 107.1 31 32.1 102.1 +4.7 45.0 115.0 +7.5

EXAMPLE 2

The effect of supplementation of a normal diet with multiple daily dosesof beta-alanine and L-histidine on the carnosine content of type I, IIA,and IIB skeletal muscle fibers of humans was assessed. The plasmaconcentration of beta-alanine in six normal subjects following theconsumption of a broth delivering approximately 40 milligrams perkilogram body weight of beta-alanine was monitored. Doses of 10 and 20milligrams per kilogram body weight of beta-alanine were also given.

The broth was prepared as follows. Fresh chicken breast (skinned andboned) was finely chopped and boiled for fifteen minutes with water (1liter for every 1.5 kg of chicken). Residual chicken meat was removed bycourse filtration. The filtrate was flavored by the addition of carrot,onion, celery, salt, pepper, basil, parsley and tomato puree, andreboiled for a further fifteen minutes and then cooled from finalfiltration though fine muslin at 4° C. The yield from 1.5 kilograms ofchicken and one liter of water was 890 mL of broth. A portion of thestock was assayed for the total beta-alanyl-dipeptide content (e.g.,carnosine and anserine) and beta-alanine. Typical analyses were:

total beta-alanyl-dipeptides 74.5 mM free beta-alanine 5.7 mM

The six male test subjects were of normal health and between 25-53 yearsof age, as shown in Table 4. The study commenced after an overnight fast(e.g., a minimum of 12 hours after the ingestion of the last meatcontaining meal). Subjects were given the option to consume a smallquantity of warm water prior to the start of the study. Catheterizationwas begun at 08:30 and the study started at 09:00.

As a control, 8 milliliters per kilogram body weight of water wasingested (e.g., 600 mL in a subject weighing 75 kilograms).

In one session, 8 milliliters per kilogram body weight of brothcontaining approximately 40 milligrams per kilogram body weight ofbeta-alanine (e.g., in the form of anserine and carnosine) was ingested.For a subject weighing 75 kilograms this amounted to the ingestion of600 milliliters of broth containing 3 grams of beta-alanine. In anothersession, 3 milliliters per kilogram body weight of a liquid containingthe test amount of beta-alanine with an additional 5 milliliters perkilogram body weight of water was ingested. In all sessions, subjectsadditionally consumed a further 8 milliliters per kilogram body weightof water (in 50 mL portions) during the period of 1 to 2 h afteringestion. A vegetarian pizza was provided after 6 hours. An ordinarydiet was followed after 8 hours.

2.5 milliliter venous blood samples were drawn through an indwellingcatheter at 10 minute intervals for the first 90 minutes and then after120, 180, 240 and 360 minutes. The blood samples were dispensed intotubes containing lithium-heparin as anti-coagulant. The catheter wasmaintained by flushing with saline. Plasma samples were analyzed by HPLCaccording to the method described in Jones & Gilligan (1983) J.Chromatoqr. 266:471-482 (1983).

Table 4 summarizes the allocation of treatments during the beta-alanineabsorption study. The estimated equivalent doses of beta-alanine arepresented in Table 3.

TABLE 4 Broth β-ala β-ala β-ala β-ala 40 0 10 20 40 Carnosine Age Weightmg/kg mg/kg mg/kg mg/kg mg/kg 20 mg/kg Subject yrs kg bwt bwt bwt bwtbwt bwt 1 53 76 ✓ ✓ ✓ ✓ 2 33 60 ✓ ✓ ✓ 3 29 105  ✓ ✓ ✓ ✓ 4 31 81 ✓ ✓ ✓ ✓5 30 94 ✓ ✓ ✓ ✓ 6 25 65 ✓ ✓ ✓ ✓

Plasma concentration curves following each treatment are depictedgraphically in FIG. 8. Mean results of the administration ofbeta-alanine, broth, or carnosine according to the treatments schedulein Table 4. Plasma beta-alanine was below the limit of detection in allsubjects on the control treatment. Neither carnosine or anserine weredetected in plasma following ingestion of the chicken broth or any ofthe other treatments. Ingestion of the broth resulted in a peakconcentration in plasma of 427.9 (SD 161.8) μM. Administration ofcarnosine equivalent to 20 milligrams per kilogram body weight ofbeta-alanine in one test subject resulted in an equivalent increase inthe plasma beta-alanine concentration.

Administration of all treatments except control resulted in an increasein the plasma taurine concentration. The changes in taurineconcentration mirrored closely those of beta-alanine. Administration ofbroth, a natural food, caused the an equivalent increase in plasmataurine, indicating that such a response is occurring normally followingthe ingestion of most meals.

EXAMPLE 3

The effect of administration of three doses of 10 milligrams perkilogram body weight of beta-alanine per day (i.e., administered in themorning, noon, and at night) for seven days on the plasma concentrationprofiles of beta-alanine and taurine were investigated. The plasmaconcentration profiles following administration of 10 milligrams perkilogram body weight of beta-alanine were studied in three subjects atthe start and end of a seven day period during which they were giventhree doses of the beta-alanine per day.

Three male subjects of normal health, aged between 33-53 years werestudied. Test subjects received three doses per day of 10 milligrams perkilogram body weight of beta-alanine for eight days. In two subjects,this was followed by a further 7 days (days 9-15) when three doses of 20milligrams per kilogram body weight per day were given. Subjectsreported at 8 am to the blood collection laboratory on days 1 (prior toany treatment given), 8 and 15 following an overnight fast. Subjectswere asked not to consume any meat containing meal during the 12 hourspreceeding the study. On each of these three test days subjects werecatheterized and an initial blood sample taken when the beta-alanine wasadministered at or close to 9 am, 12 noon, and 3 pm. Blood samples weredrawn after 30, 60, 120 and 180 minutes, and analyzed for changes in theplasma concentration of beta-alanine and taurine. 24 hour urine sampleswere collected over each day of the study and analyzed by HPLC todetermine the excretion of beta-alanine and taurine. The treatments aresummarized in Table 5.

TABLE 5 Treatment Day Day 1 Day 8 Day 15 beta-alanine 10 mg/kg bwt 10mg/kg bwt 20 mg/kg bwt 1 ✓ ✓ ✓ 2 ✓ ✓ ✓ 3 ✓ ✓

The plasma beta-alanine concentrations are summarized in FIG. 9. Eachdose resulted in a peak beta-alanine concentration at one-half hour orone hour after ingestion followed by a decline to a 0-10 micromolarbasal level at three hours, just prior to administration of the nextdose. The response on day 8 of the treatment tended to be less than onday 1 as indicated by the area under the plasma concentration curve.

EXAMPLE 4

The effect of administration of three doses of 40 milligrams perkilogram body weight of beta-alanine per day (i.e., administered in themorning, noon, and at night) for 2 weeks on the carnosine content ofmuscle, and isometric endurance at 66% of maximal voluntary contractionforce.

Six normal male subjects, aged 25 to 32 years, that did not haveevidence of metabolic or muscle disease were recruited into the study.The subjects were questioned regarding their recent dietary andsupplementary habits. None of subjects was currently taking supplementscontaining creatine, or had done so in recent testing supplementationprocedures. The physical characteristics of the test subjects aresummarized in Table 6.

TABLE 6 Subject Age (years) Weight (kg) 1 29  78 2 31  94 3 29 105 4 25 65 5 31  81 6 25  75 7 53  76

Two days before treatment, a preliminary determination of maximalvoluntary (isometric) contraction force (MVC) of knee extensors with thesubject in the sitting position was carried out. MVC was determinedusing a Macflex system with subjects motivated by an instantaneousvisual display of the force output. For each subject, two trials werecarried out to determine endurance at 66% MVC sustained until the targetforce could no longer be maintained despite vocal encouragement. Thisfirst contraction was subsequently followed by a rest period of 60seconds, with the subject remaining in the isometric chair. After therest period, a second contraction was sustained to fatigue. Following asecond rest of 60 seconds, a third contraction to fatigue wasundertaken.

One day before treatment, the subjects reported to the isometric testlaboratory between 8 and 10 am. MVC was determined and endurance at 66%MVC over three contractions with 60 second rest intervals, as describedabove, was determined. Measurements were determined using the subject'sdominant leg. A biopsy of the lateral portion of the vastus lateraliswas taken again from the dominant leg.

On day 1 of the treatment study, subjects reported to the blood samplinglaboratory at 8 am following an overnight fast and a minimum of 12 hourssince the last meat containing meal. Following catheterization and abasal blood sample, each subject followed the supplementation and bloodsampling protocol described in Example 3. A dose of 10 milligrams perkilogram body weight of beta-alanine was administered at time 0 (9 am),3 hours, and 6 hours.

On days 2-15, subjects continued to take three doses of 10 milligramsper kilogram body weight of beta-alanine.

In the morning of day 14, post-treatment isometric exercise tests wereconducted on the dominant leg to determine MVC and endurance at 66% MVCrelative to the 66% MVC measured on the day prior to treatment. In theafternoon, a muscle biopsy was taken of the vastus lateralis from closeto the site of the biopsy taken on the day before treatment.

On day 15, the procedures followed on day 1 were repeated to determineany overall shift in the plasma concentration profile of beta-alanineand taurine over the 15 days of supplementation. Mean changes in plasmabeta-alanine over 9 hours following the oral ingestion of 10 milligramsper kilogram body weight of beta-alanine at 0, 3 and 6 hours on days 1and 15 when dosing at 3×10 milligrams per kilogram body weight per dayare shown in FIG. 10.

One additional test subject (number 7) followed the study, taking threedoses 10 milligrams per kilogram body weight for 7 days followed bythree doses of 20 milligrams per kilogram body weight for 7 days. Nomuscle biopsies were taken from this test subject.

There was no apparent change in the muscle carnosine content in themuscle of the six subjects biopsied. Changes in plasma taurineconcentrations in the six subjects mirrored those of beta-alanine, asnoted in Example 2.

Values from the MVC and endurance at 66% MVC measurements one day beforetreatment and after 14 days after treatment with three doses of 10milligrams per kilogram body weight of beta-alanine are listed in Table7. The mean endurance time at 66% MVC increased in 5 of the 6 subjects.An increase was also seen in subject 7 taking the higher dose.

TABLE 7 time @ time @ time @ Total MVC MVC 66% MVC 66% MVC 66% MVCContraction 1st try 2nd try 1st 2nd 3rd Time Subject N N seconds secondsseconds seconds Pre 1 784.5 821.9 48.53 29.03 23.78 100.83 2 814.4 886.248.40 26.03 16.90 91.33 3 984.9 970.4 38.15 26.03 16.78 80.95 4 714.6740.4 89.03 56.15 45.65 190.83 5 1204.8 1217.2 37.65 27.64 21.53 86.83 6722.4 716.8 46.78 29.40 21.90 98.08 Pre mean 870.9 892.1 51.4 32.4 24.3108.1 Pre SD 190.6 184.6 19.1 11.7 10.8 41.2 Post 1 895.6 908.0 47.0830.38 24.03 101.48 2 832.2 908.0 46.65 31.28 18.40 96.33 3 973.7 952.242.65 25.03 16.03 83.70 4 814.1 863.9 114.40 64.28 48.53 227.20 5 1246.61233.0 42.03 22.78 19.40 84.20 6 760.8 773.3 52.28 31.53 25.95 109.73Post mean 920.5 939.7 57.5 34.2 25.4 117.1 Post SD 175.7 156.0 28.1 15.211.9 54.9 Subject 7 Pre 858.18 861.54 54.0 Post 792.54 851.41 62.0

Other embodiments are within the claims.

What is claimed is:
 1. A composition consisting essentially of: apeptide source including beta-alanine; between about 39 and about 99percent by weight of a carbohydrate; and up to about 60 percent byweight of water, wherein the composition includes between about 1 andabout 20 percent by weight of the beta-alanine.
 2. The composition ofclaim 1, wherein the peptide source includes L-histidine and thecomposition includes between about 1 and about 20 percent by weight ofL-histidine.
 3. A composition consisting essentially of: a peptidesource including beta-alanine; between about 1 and about 98 percent byweight of a creatine source; and up to about 97 percent by weight ofwater, wherein the composition includes between about 1 and about 98percent by weight of the beta-alanine.
 4. The composition of claim 3,wherein the peptide source includes L-histidine and the compositionincludes between about 1 and about 98 percent by weight of L-histidine.5. A method of increasing the anaerobic working capacity of a tissuecomprising: providing an amount of beta-alanine to blood or blood plasmaeffective to increase beta-alanylhistidine dipeptide synthesis in atissue; providing an amount of L-histidine to the blood or blood plasmaeffective to increase beta-alanylhistidine dipeptide synthesis; andexposing the tissue to the blood or blood plasma, whereby theconcentration of beta-alanylhistidine is increased in the tissue.
 6. Themethod of claim 1, further comprising increasing a concentration ofcreatine in the tissue.
 7. The method of claim 1, wherein the providingsteps include ingestion of a composition including the amount ofbeta-alanine and the amount of L-histidine.
 8. The method of claim 1,wherein the providing step includes infusion of a composition includingthe amount of beta-alanine and the amount of L-histidine.
 9. The methodof claim 1, further comprising increasing a concentration of insulin inthe blood or blood plasma.
 10. The method of claim 1, wherein the tissueis a skeletal muscle.
 11. The method of claim 1, wherein the tissue is ahuman tissue.
 12. The method of claim 1, wherein the tissue is an animaltissue.